The PI3K subunits, P110? and P110? are potential targets for overcoming P-gp and BCRP-mediated MDR in cancer.
ABSTRACT: BACKGROUND:PI3K/AKT is a vital signaling pathway in humans. Recently, several PI3K/AKT inhibitors were reported to have the ability to reverse cancer multidrug resistance (MDR); however, specific targets in the PI3K/AKT pathways and the mechanisms associated with MDR have not been found because many of the inhibitors have multiple targets within a large candidate protein pool. AKT activation is one presumed mechanism by which MDR develops during cancer treatment. METHODS:The effects of inhibiting PI3K 110? and 110? by BAY-1082439 treatment and CRISPR/Cas9 knockout were examined to determine the possible functions of BAY-1082439 and the roles of PI3K 110? and 110? in the reversal of MDR that is mediated by the downregulation of P-gp and BCRP. Inhibition of AKT with GSK-2110183 showed that the downregulation of P-gp and BCRP is independent of generalized AKT inactivation. Immunofluorescence, immunoprecipitation, MTT, flow cytometry and JC-1 staining analyses were conducted to study the reversal of MDR that is mediated by P-gp and BCRP in cancer cells. An ATPase assay and a structural analysis were also used to analyze the potential mechanisms by which BAY-1082439 specifically targets PI3K 110? and 110? and nonspecifically influences P-gp and BCRP. RESULTS:By inhibiting the activation of the PI3K 110? and 110? catalytic subunits through both the administration of BAY-1082439 and the CRISPR/Cas9 deletion of Pik3ca and Pik3cb, the ATP-binding cassette transporters P-gp/ABCB1 and BCRP/ABCG2 were downregulated, thereby reestablishing the drug sensitivity of human epidermoid carcinoma and non-small cell lung cancer (NSCLC) MDR cells. Inhibition of AKT did not reverse the MDR mediated by P-gp or BCRP. The ABC family proteins and AKT may play MDR-enhancing roles independently. CONCLUSIONS:The reversal of the dual functions of ABC-transporter-mediated and AKT-activation-enhanced MDR through the inhibition or knockout of PI3K 110? or 110? promises to improve current strategies based on combined drug treatments to overcome MDR challenges.
Project description:ATP binding cassette (ABC) transporters, such as P-gp, BCRP and MRP1, can increase efflux of clinical chemotherapeutic agents and lead to multi-drug resistance (MDR) in cancer cells. While the discovery and development of clinically useful inhibitors has proved elusive to date, this molecular target nevertheless remains a promising strategy for addressing and potentially overcoming MDR. In a search for new classes of inhibitor, we used fluorescent accumulation and efflux assays supported by cell flow cytometry and MDR reversal assays, against a panel of sensitive and MDR human cancer cell lines, to evaluate the marine sponge co-metabolites 1-12 as inhibitors of P-gp, BCRP or MRP1 initiated MDR. These studies identified and characterized lamellarin O (11) as a selective inhibitor of BCRP mediated drug efflux. A structure-activity relationship analysis inclusive of the natural products 1-12 and the synthetic analogues 13-19, supported by in silico docking studies, revealed key structural requirements for the lamellarin O (11) BCRP inhibitory pharmacophore.
Project description:<h4>Background and purpose</h4>Multidrug resistance (MDR), usually mediated by overexpression of efflux transporters such as P-gp, ABCG2 and/or MRP1, remains a major obstacle hindering successful cancer chemotherapy. There has been great interest in the development of inhibitors towards these transporters to circumvent resistance. However, since the inhibition of transporter is not specific to cancer cells, a decrease in the cytotoxic drug dosing may be needed to prevent excess toxicity, thus undermining the potential benefit brought about by a drug efflux inhibitor. The design of potent MDR modulators specific towards resistant cancer cells and devoid of drug-drug interactions will be needed to effect MDR reversal.<h4>Experimental approach</h4>Recent evidence suggests that the PTEN/PI3K/Akt pathway may be exploited to alter ABCG2 subcellular localization, thereby circumventing MDR. Three PPAR? agonists (telmisartan, pioglitazone and rosiglitazone) that have been used in the clinics were tested for their effect on the PTEN/PI3K/Akt pathway and possible reversal of ABCG2-mediated drug resistance.<h4>Key results</h4>The PPAR? agonists were found to be weak ABCG2 inhibitors by drug efflux assay. They were also shown to elevate the reduced PTEN expression in a resistant and ABCG2-overexpressing cell model, which inhibit the PI3K-Akt pathway and lead to the relocalization of ABCG2 from the plasma membrane to the cytoplasma, thus apparently circumventing the ABCG2-mediated MDR.<h4>Conclusions and implications</h4>Since this PPAR?/PTEN/PI3K/Akt pathway regulating ABCG2 is only functional in drug-resistant cancer cells with PTEN loss, the PPAR? agonists identified may represent promising agents targeting resistant cells for MDR reversal.
Project description:Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1). Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers.
Project description:The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.
Project description:Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of ABC transporter proteins, including P-170 glycoprotein (P-gp), and breast cancer resistance protein (BCRP, ABCG2) have been implicated in this multi-drug resistance (MDR). These ABC transporters are ATP-dependent efflux proteins. We have recently shown that nitric oxide (NO) inhibits the ATPase activities of P-gp, resulting in a significant enhancement of drug accumulation and the reversal of multi-drug resistance in NCI/ADR-RES cells, a P-gp-overexpressing human MDR cell line. In this study, we used [O<sup>2</sup>-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1?yl]-diazene-1-ium-1-2-diolate] (JS-K), a tumor-specific NO-donor to study the reversal of drug resistance in both P-gp- and BCRP-overexpressing human tumor cells. We report here that while JS-K was extremely effective in reversing adriamycin resistance in the P-gp-overexpressing tumor cells (NCI/ADR-RES); it was significantly resistant to BCRP-overexpressing (MCF-7/MX) tumor cells, suggesting that JS-K may be a substrate for BCRP. Using another NO-donor (DETNO), we show that NO directly inhibits the ATP activities of BCRP, inducing significant increases in the accumulations of both Hoechst 33342 dye and topotecan, substrates for BCRP. Furthermore, NO treatment significantly reversed topotecan and mitoxantrone resistance to MCF-7/MX tumor cells. Molecular docking studies indicated that while DETNO and JS-K bind to ATP binding site in both ABC proteins, binding score was significantly reduced, compared to the ATP binding. Our results indicate that appropriately designed NO donors may find success in reversing multidrug resistance in the clinic.
Project description:Previously, we reported sipholenol A, a sipholane triterpenoid from the Red Sea sponge Callyspongia siphonella, as a potent reversal of multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp). Through extensive screening of several related sipholane triterpenoids that have been isolated from the same sponge, we identified sipholenone E, sipholenol L and siphonellinol D as potent reversals of MDR in cancer cells. These compounds enhanced the cytotoxicity of several P-gp substrate anticancer drugs, including colchicine, vinblastine and paclitaxel, and significantly reversed the MDR-phenotype in P-gp-overexpressing MDR cancer cells KB-C2 in a dose-dependent manner. Moreover, these three sipholanes had no effect on the response to cytotoxic agents in cells lacking P-gp expression or expressing MRP1 (ABCC1) or MRP7 (ABCC10) or breast cancer resistance protein (BCRP/ABCG2). All three sipholanes (IC(50) >50 ?M) were not toxic to all the cell lines that were used. [(3)H]-Paclitaxel accumulation and efflux studies demonstrated that all three triterpenoids time-dependently increased the intracellular accumulation of [(3)H]-paclitaxel by directly inhibiting P-gp-mediated drug efflux. Sipholanes also inhibited calcein-AM transport from P-gp-overexpressing cells. The Western blot analysis revealed that these three triterpenoids did not alter the expression of P-gp. However, they stimulated P-gp ATPase activity in a concentration-dependent manner and inhibited the photolabeling of this transporter with its transport substrate [(125)I]-iodoarylazidoprazosin. In silico molecular docking aided the virtual identification of ligand binding sites of these compounds. In conclusion, sipholane triterpenoids efficiently inhibit the function of P-gp through direct interactions and may represent potential reversal agents for the treatment of MDR.
Project description:The emergence of multidrug resistance (MDR) in the clinic is a significant problem for a successful treatment of human cancers. Overexpression of various ABC transporters (P-gp, BCRP and MRP's), which remove anticancer drugs in an ATP-dependent manner, is linked to the emergence of MDR. Attempts to modulate MDR have not been very successful in the clinic. Furthermore, no single agent has been found to significantly inhibit their functions to overcome clinical drug resistance. We have previously shown that nitric oxide (<sup>●</sup>NO) inhibits ATPase functions of ABC transporters, causing reversal of resistance to clinically active anticancer drugs. In this study, we have used cytotoxicity and molecular docking studies to show that NCX4040, a nitric oxide donor related to aspirin, inhibited the functions of ATPase which resulted in significant reversal of resistance to both adriamycin and topotecan in P-gp- and BCRP-expressing human cancer cell lines, respectively. We also used several other cytotoxic nitric oxide donors, e.g., molsidomine and S-nitroso glutathione; however, both P-gp- and BCRP-expressing cells were found to be highly resistant to these NO-donors. Molecular docking studies showed that NCX4040 binds to the nucleotide binding domains of the ATPase and interferes with further binding of ATP, resulting in decreased activities of these transporters. Our results are extremely promising and suggest that nitric oxide and other reactive species delivered to drug resistant tumor cells by well-designed nitric oxide donors could be useful in sensitizing anticancer drugs in multidrug resistant tumors expressing various ABC transporters.
Project description:Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) is an ATP-binding cassette (ABC) transporter identified as a molecular cause of multidrug resistance (MDR) in diverse cancer cells. BCRP physiologically functions as a part of a self-defense mechanism for the organism; it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract, as well as through the blood-brain, placental, and possibly blood-testis barriers. BCRP recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules relatively new in clinical use. Thus, BCRP expression in cancer cells directly causes MDR by active efflux of anticancer drugs. Because BCRP is also known to be a stem cell marker, its expression in cancer cells could be a manifestation of metabolic and signaling pathways that confer multiple mechanisms of drug resistance, self-renewal (stemness), and invasiveness (aggressiveness), and thereby impart a poor prognosis. Therefore, blocking BCRP-mediated active efflux may provide a therapeutic benefit for cancers. Delineating the precise molecular mechanisms for BCRP gene expression may lead to identification of a novel molecular target to modulate BCRP-mediated MDR. Current evidence suggests that BCRP gene transcription is regulated by a number of trans-acting elements including hypoxia inducible factor 1?, estrogen receptor, and peroxisome proliferator-activated receptor. Furthermore, alternative promoter usage, demethylation of the BCRP promoter, and histone modification are likely associated with drug-induced BCRP overexpression in cancer cells. Finally, PI3K/AKT signaling may play a critical role in modulating BCRP function under a variety of conditions. These biological events seem involved in a complicated manner. Untangling the events would be an essential first step to developing a method to modulate BCRP function to aid patients with cancer. This review will present a synopsis of the impact of BCRP-mediated MDR in cancer cells, and the molecular mechanisms of acquired MDR currently postulated in a variety of human cancers.
Project description:Lung cancer is the leading cause of cancer-related deaths worldwide and remains the most prevalent. Interplay between PI3K/AMPK/AKT and MAPK pathways is a crucial effector in lung cancer growth and progression. These signals transduction protein kinases serve as good therapeutic targets for non-small cell lung cancer (NSCLC) which comprises up to 90% of lung cancers. Here, we described whether 21?-Methylmelianodiol (21?-MMD), an active triterpenoid derivative of Poncirus trifoliate, can display anticancer properties by regulating these signals and modulate the occurrence of multidrug resistance in NSCLC cells. We found that 21?-MMD inhibited the growth and colony formation of lung cancer cells without affecting the normal lung cell phenotype. 21?-MMD also abrogated the metastatic activity of lung cancer cells through the inhibition of cell migration and invasion, and induced G0/G1 cell cycle arrest with increased intracellular ROS generation and loss of mitochondrial membrane integrity. 21?-MMD regulated the expressions of PI3K/AKT/AMPK and MAPK signaling which drove us to further evaluate its activity on multidrug resistance (MDR) in lung cancer cells by specifying on P-glycoprotein (P-gp)/MDR1-association. Employing the established paclitaxel-resistant A549 cells (A549-PacR), we further found that 21?-MMD induced a MDR reversal activity through the inhibition of P-gp/MDR1 expressions, function, and transcription with regained paclitaxel sensitivity which might dependently correlate to the regulation of PI3K/mTOR signaling pathway. Taken together, these findings demonstrate, for the first time, the mechanistic evaluation in vitro of 21?-MMD displaying growth-inhibiting potential with influence on MDR reversal in human lung cancer cells.
Project description:Multidrug resistance (MDR) in cancer is one of the main limitations for chemotherapy success. Numerous mechanisms are behind the MDR phenomenon wherein the overexpression of the ATP-binding cassette (ABC) transporter proteins P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 1 (MRP1) is highlighted as a prime factor. Natural product-derived compounds are being addressed as promising ABC transporter modulators to tackle MDR. Flavonoids and terpenoids have been extensively explored in this field as mono or dual modulators of these efflux pumps. Nitrogen-bearing moieties on these scaffolds were proved to influence the modulation of ABC transporters efflux function. This review highlights the potential of semisynthetic nitrogen-containing flavonoid and terpenoid derivatives as candidates for the design of effective MDR reversers. A brief introduction concerning the major role of efflux pumps in multidrug resistance, the potential of natural product-derived compounds in MDR reversal, namely natural flavonoid and terpenoids, and the effect of the introduction of nitrogen-containing groups are provided. The main modifications that have been performed during last few years to generate flavonoid and terpenoid derivatives, bearing nitrogen moieties, such as aliphatic, aromatic and heterocycle amine, amide, and related functional groups, as well as their P-gp, MRP1 and BCRP inhibitory activities are reviewed and discussed.